Bone marrow (BM) transplantation was the first and continues to be the most successful example of a stem cell-based therapy. Nevertheless, there remains a constant, critical shortage of stem cells to meet the needs of patients suffering from hematological or other diseases requiring BM transplantation. This shortage is due to a lack of sufficient numbers of immunologically compatible donors and the limited numbers of hematopoietic stem cells (HSCs) contained within a donor product, especially in umbilical cord blood transplantation.
HSC transplantation (HSCT) was the first successful example of a stem cell-based treatment. The systematic application of HSCT, however, has been limited by the availability of matching donors, by administration of immunosuppressive drugs to prevent transplant rejection, and by the limited HSC number/availability.
Direct programming of HSCs from reprogramming differentiated cells, whether from the individual patient or a suitable donor, offers an exciting alternative to overcome these limitations. This reprogramming of differentiated cells to HSCs also avoids ethical issues associated with the use of HSCs derived from embryonic stem cells (ESCs) and circumvents complete induced pluripotent stem cell (iPSC) reprogramming to pluripotency with subsequent directed differentiation to hematopoietic cells. Indeed, the directed differentiation of either ESCs or iPSCs to bona fide HSCs remains elusive. Direct programming of HSCs could provide a universal and unlimited source material for cell replacement therapy of hematological diseases.
Reprogramming cell identity holds great promise for biomedicine as a major source of patient-specific cell-types for transplantation-based therapies. While reprogramming was achieved towards pluripotent and some differentiated cell fates, the direct programming of multipotent adult stem cells, such as HSCs remain to be accomplished.
HSCs continuously replenish all blood cell lineages. Their hallmark property is the ability to strike a balance between self-renewal and differentiation to form mature blood. The transcriptional regulatory network of HSCs is just starting to be addressed on a global scale. Genome-wide binding maps of transcription regulators and gain-of-function screening approaches recently have provided global insight on the combinatorial transcriptional control of HSCs (Wilson et al., Cell Stem Cell 7:532-44, 2010; Deneault et al., Cell 137: 369-79, 2009). Seminal experiments by Yamanaka and colleagues showed that retroviral-mediated expression of Oct4, Sox2, c-Myc and Klf4 could drive mouse and human fibroblasts into an iPSC state (Takahashi et al., Cell 126: 663-76, 2006; Takahashi et al., Cell 131: 861-72, 2007). These pioneering studies have illustrated the importance of a limited combination of transcription factors for the induction of pluripotency. Later studies have shown that certain factors could be replaced with small molecules and the direct conversion towards other cell identities (Hanna et al., Cell 143: 508-25, 2010). In this way, for example, the transcription factors C/EBPα/β and PU.1 were found to induce a macrophage fate in lymphoid cells and fibroblasts (Xie et al., Cell 117: 663-76, 2004; Feng et al., Proc. Natl. Acad. Sci. USA 105: 6057-62, 2008); Ascl1, Brn2/Pou3f2 and Mytl1 to induce neuronal identity in fibroblasts (Vierbuchen et al., Nature 463:1035-41, 2010); Gata4, Mef2c and Tbx5 to induce fibroblasts to cardiomyocytes (Leda et al., Cell 142: 375-86, 2010); Ngn3, Pdx1, and Mafa reprogram pancreatic exocrine cells to beta cells (Zhou et al., Nature 425:627-632, 2008); and Gata4, Hnf1a and FoxA3 induce hepatocyte-like cells from mouse fibroblasts (Huang et al., Nature 475:386-391, 2011). In addition, it recently was reported that overexpression of Oct4 together with specific cytokine treatment can direct fibroblasts to a myeloid/erythroid progenitor cell fate (Szabo et al, Nature 468: 521-6, 2010). It is unclear, however, if Oct4 acts by inducing de-differentiation or by mimicking the action of the family member Oct1 which is expressed in hematopoietic tissues. The hematopoietic progenitors generated do not retain self-renewal and the same degree of multipotency as HSCs, e.g., hematopoietic progenitors do not give rise to lymphoid cells.
Collectively, these direct reprogramming results raise the question of whether there is a particular transcription factor signature that can be used for programming adult somatic stem cells that self-renew and are able to differentiate in all lineages of the hemato-lymphoid system. To date, direct programming of differentiated cells to bona fide HSCs or primitive hematopoietic progenitors has not been demonstrated.
Thus, the art to date does not disclose methods for the direct programming of HSCs. Accordingly, a strong need in the art exists for a method of programming differentiated cells into HSCs. The following disclosure describes the specifics of such a method.